Recent studies have demonstrated new complexities in both innate and adaptive components of the immune response, and suggest that successful induction of antitumor responses may depend heavily on how effectively appropriate innate and adaptive components communicate with each other. We have used transplantable mouse renal, autochthonous mammary, and transplantable lung carcinoma models to demonstrate that the systemic administration of the combinations of IL-12/pulse IL-2 and IL-18/IL-2 yield enhanced IFN-gamma dependent, Fas dependent antitumor effects against even well-established metastatic cancers. The effects of both cytokine combinations are rapidly initiated by a mechanism that includes the upregulation of antiangiogenic and apoptosis-associated genes in the tumor site and the destruction of tumor vasculature-associated endothelial cells. These promising results have resulted in the initiation of a phase I/II trial of IL-12/pulse IL-2 at the NCI, and successful negotiations for the initiation of a phase I trial of IL-18/IL-2. However, due to the weak immunogenicity of human cancers, the coordinate engagement of professional antigen-presenting cells and antigen-specific cell-mediated effector cells may provide additional advantages for induction of effective anti-tumor responses. In this regard we hypothesized that the enhanced differentiation and function of dendritic cells through CD40 engagement, combined with IL-2 administration to stimulate T cell expansion, would act coordinately to enhance both the antigen-presenting and T cell components of the adaptive immune response against cancer. In mice bearing orthotopic metastatic renal cell carcinoma, only the combination of anti-CD40 and IL-2, but neither agent administered alone, induced complete regression of metastatic tumor and specific immunity to subsequent re-challenge in the majority of treated mice. IL-2 treatment alone enhanced the expression of CD40 on dendritic cells and the combination of anti-CD40 and IL-2 resulted in significant increases in dendritic cells and CD8+ T cells in advanced tumor-bearing mice. The anti-tumor effects of anti-CD40 and IL-2 were dependent on CD8+ T cells, interferon-gamma, IL-12 p40 and Fas ligand. These events culminated in enhanced tumor-specific T cell responsiveness and complete regression of metastatic cancer. These findings provide a basis for further studies to determine the effectiveness of anti-CD40 in the presence or absence of various T cell stimulating cytokines against various tumor types, the ability of this strategy to enhance responses to defined antigens, and the mechanisms by which these effects can occur. In addition, to the strategies described above for developing logical combinations of cytokines, we are also studying new approaches to maximize their therapeutic impact, in a setting of acceptable toxicity. In this regard we have applied a technique termed hydrodynamic gene delivery to administer non-viral, DNA plasmids encoding a variety of cytokines (IL-2, IL-12, (IL-15, FLT3L) to mice. These results of these experiments have shown that the hydrodynamic injection of appropriate cytokine-encoding DNA plasmids results in high and sustained levels of the cytokine, expected effects on innate or adaptive immune components and antitumor efficacy in several mouse models. Overall, these results provide us with new approaches for utilizing cytokines for cancer treatment, and have the additional practical advantage.